On-going studies of the collaboration scenarios between T cells and antigen-presenting cells (APCs) foresaw dramatic changes which have revealed some therapeutic targets and medicinal potentials in certain regards. For example, it has been shown that the specialized and dynamic molecular machinery, present in the tight junction between a T cell and an APC, regulates immunological responses (Dustin, 2002; Grakoui et al., 1999; Qi et al., 2001). It has also been inferred that the machinery, termed the immunological synapse, correlates with a high degree of intercellular communication controlling disparate biological process (Davis and Dustin, 2004). A number of molecules have been confined at the immunological synapse to ensure their interim expression and interaction at the right time and place thus the sum and integration of signals are relevant to evoke appropriate T cell responses. Within this limited and—μm-sized area is full of interacting molecules, of which CTLA-4 has been identified to be responsible for inhibiting T cell responses in a T cell receptor (TCR)-dependent manner (Chikuma and Bluestone, 2002; Egen and Allison, 2002).
Human CTLA-4 was mapped to band q33 of chromosome 2 and was classified into a group of immunomodulating receptors, collectively termed as CD28 superfamily (Sharpe and Freeman, 2002). As shown in FIG. 1, the complete cDNA sequence of human CTLA-4 (isoform A) has the Genbank accession number L15006 and the structure has the accession number 1AH1 in the Molecular Modeling DataBase (MMDB) of NCBI's structure database. The region of amino acids −35 to 0 is the singal peptide; 1-126 is the extracellular V-like domain; 123-151 is the transmembrane domain; and 152-188 is the cytoplasmic domain. It is well established that two members of this superfamily, CD28 and CTLA-4, have opposing functions and that CTLA-4 represents one of the major inhibitory receptors involved in co-stimulatory pathways regulating both humoral and cellular immune responses (Krummel and Allison, 1996; Linsley et al., 1991; Pioli et al., 2000). A majority of studies indicate that CD28 provides direct enhancement signals, including up-regulation/stabilization of cytokine gene transcription, improved cell survival, lowered threshold for activation, and cytoskeletal effects; however, information on the function of CTLA-4 is much less clear. Thus far, the most compelling evidence for an inhibitory role of CTLA-4 is derived from the deficient knockout mice (CTLA-4−/−) (Tivol et al., 1995). These mice suffer from a fatal T-cell lymphoproliferative disorder with splenomegaly, lymphoadenopathy and hyper-responsive infiltration in several organs including heart that become apparent by four weeks after birth. This fetal disorder is presumably due to reactivities to multiple self-antigens, since the expression of a single transgenic TCR prevents this disease. The TCR-dependent activation in these knockout mice appears to require CD28 costimulation, because CTLA-4−/−CD28−/− mice do not suffer the lymphoproliferative disease. Likewise, treatment of these mice perinatally with soluble CTLA-4-Ig, which competes ligands access of cell surface CTLA-4, prevents such a disease effectively. Nonetheless, the mechanism of CTLA-4 action is still unclear, with no obvious central theme.
Conceptually, the interaction of CD28 on the lymphocyte with B7 proteins on the APC provides a necessary costimulatory second signal for a T cell to be able to fully respond to an antigen. The original family members in the pathway consist of two B7 ligands—CD80 (B7-1) and CD86 (B7-2), which have specificities towards the two receptors—CD28 and CTLA-4. CD28 is constitutively expressed on the surface of T cells whereas CTLA-4 surface expression is rapidly up-regulated to a limited extent following T cell activation. The kinetics of expression of CD80 and CD86 also differ. CD86 is constitutively expressed on interdigitating dendritic cells, Langerhans cells, peripheral blood dendritic cells, memory B cells and germinal center B cells. Furthermore, CD86 is expressed at low levels on monocytes, but its rapid up-regulation through IFN-γ stimulation has led to the hypothesis that CD86 functions primarily in initiating an immune response. On the other hand, CD80, being expressed later in time, may serve to amplify or regulate the response. Newly identified family members of the related molecules include: the inducible costimulatory molecule (ICOS), program death 1 (PD-1) receptor, B and T lymphocyte attenuator (BTLA), B7-H1, B7-H2, B7-H3, B7-H4, PD-1 ligand 1 (PD-L1) and PD-L2 (Wang and Chen, 2004). The novel interactions among these new family members underscore additional complexity of this costimulatory pathway in mounting an appropriate immune response.
A shorter soluble form of CTLA-4 lacking the transmembrane region has been achieved from RT-PCR cloning of non-activated T cells in animals as well as humans (Magistrelli et al., 1999; Oaks et al., 2000). Soluble CTLA-4 (sCTLA-4, CTLA-4 isoform B; FIG. 1) seems to be a fully functional CD80 and CD86 receptor, thus likely to affect T-cell responses in a paracrine manner. Furthermore, immunoreactive sCTLA-4 can be detected in the serum of 14/64 healthy subjects. In addition, the presence of high concentration of sCTLA-4 was observed in sera of patients with autoimmune thyroid diseases such as Graves' disease (Oaks and Hallett, 2000). Finally, recent reports show that sCTLA-4 levels are augmented in patients with autoimmune diseases, such as type-1 diabetes (Nistico et al., 1996), diffuse cutaneous systemic sclerosis (Sato et al., 2004), systemic lupus erythematosus (Wong et al. (2005) Rheumatology 44:989), and allergic asthma (Wong et al. (2005) Clin. Exp. Immunol. 141:122). It has been shown that activated T cells suppress sCTLA-4 mRNA expression and express preferentially the membrane-bound, full-length CTLA-4 (flCTLA-4) mRNA (Gough et al., 2005). Thus the ratio of sCTLA-4 to flCTLA-4 may have an important role in the regulation of immune homeostasis. The alternate transcripts or spliced variants of sCTLA-4, which lack the transmembrane encoding regions, were first deposited in the GenBank Sequence Database in humans, mice, and rats (accession numbers U90273, U90270, and U90271) in 1997 followed by a description of the same transcript in humans, being expressed by non-stimulated human T cells. The endogenous 174-aa soluble form, designed as isoform B, can be retrieved under the accession number NP—001032720.
It is also known in the field that immune reactivity is further controlled by various types of regulatory T cells (Tregs) (Sakaguchi, 2005). Tregs can be broadly divided into two subsets, i.e., the natural Treg cells of CD4+CD25+ phenotype, which constitute 5-10% of peripheral T cells, and the stimulation-induced (or adaptive) Treg cells identified in various models of inflammation, alloreactivity, or autoimmunity (Prud'homme, 2004). Recent findings suggest that the suppressive potential of CD4+CD25+ natural Tregs to other activated effector T cells is mediated by restricting early proliferation and the anti-effector function in inflamed tissues (von Boehmer, 2005). The forkhead-family transcription factor gene FOXP3, encoding the scurfin transcriptional regulator (Genbank accession number EF534714, NCBI protein accession number ABQ15210), has been implicated in the development and function of natural Tregs (Hori et al., 2003). A FOXP3 mutation in scurfy mice results in the absence of Tregs and early death from a multi-organ inflammatory disorder similar to the CTLA-4 or TGF-β deficiency. FOXP3 was shown to function as a transcriptional repressor, targeting composite NF-AT/AP-1 sites in cytokine gene promoters and the region responsible for NF-AT inhibition was mapped to the amino terminus (Lopes et al., 2006; Wu et al., 2006).
In principle, conventional techniques to isolate this rare Treg population often involve a two-step, multiple antibody selection procedure (Miltenyi Biotec, Bergisch Gladbach, Germany; BD Biosciences Pharmingen, San Jose, Calif.). Briefly, CD4+ T lymphocytes are first preserved from not binding to a cocktail of mAbs that recognize other CD antigens expressed on erythrocytes, platelets, monocytes and peripheral leukocytes, etc. Subsequently, anti-human CD25 mAb positively selects the CD25+ cells from the enriched CD4+ cells, yielding CD4+CD25+ Treg cells. However, inevitably intermittent exposure to environmental pathogens results in traditional effector T cell activation and consequently expression of CD25 on human CD4+ T cells, making identification of the Treg population a very difficult task. Additionally, even CD4−CD8+ natural Treg cells have been reported by Xystrakis et al. (2004) Blood 104: 3294-3301, indicating that Treg is a heterogeneous population. Furthermore, although FOXP3 expression is found predominantly within the Tregs, its intracellular nuclear localization causes direct detection impossible to live cells. Therefore, the characterization and application of Treg cells have been hampered by a lack of specific molecular markers on the surface of Tregs. A more complex approach was engineered to circumvent this particular problem, in which purified CD4+CD25+ peripheral blood mononuclear cells are further activated with agents such as ionomycin, and the Tregs are isolated based on binding to CTLA-4 blocking mAb (Birebent et al., 2004). Yet an even more complicated process has evolved by using additional surface markers like CD45RA and CD127 (WO 2007/117602).
Of interest is the association and potential synergism between the suppressive function of Tregs and the CTLA-4 expression. Unusually for non-activated T cells, Tregs constitutively express CTLA-4 (Takahashi et al., 2000), and CTLA-4 blockade on the Treg by specific blocking mAb can attenuate their suppressive activity, leading to the development of autoimmune disease in vivo (Read et al., 2006). In addition, it has been observed not only that the reported CD4−CD8+ natural Tregs express CTLA-4 (Xystrakis et al. 2004) but also that CD4+CD25+ cells further purified on the basis of recycling CTLA-4 are much more potent as regarding suppression (Birebent, et al., 2004). More importantly, the fact that inducible Tregs were the dominant source of sCTLA-4 was revealed in the 2007 British Society for Immunology Congress (Ward and Barker, 2007). Together, they indicate a strong correlation between CTLA-4 expression and suppressive regulatory function, supportive of the concept that CTLA-4 is functionally relevant to Tregs.
Because Tregs, in accompany with sCTLA-4, are involved in preventing allograft rejection and graft versus host disease and exert a dominant effect in controlling autoimmunity and maintaining peripheral tolerance, specific immune therapies designed to isolate and then expand them may improve the clinical course of various T-cell mediated pathology. As CTLA-4 provides the most important attenuating costimulatory signals, it will be expected by one of skill in the art that these molecules offer new targets for immunotherapy and diagnostics.
Studies of the physiological function and practical uses of CTLA-4 became possible with the isolation of monoclonal antibodies (mAbs). The first reported mouse anti-human CTLA-4 mAb (clone 11D4) suggested that blocking CTLA-4 signaling might deliver a positive signal synergizes with that delivered by CD28 (Linsley et al., 1992). The immune-enhancing nature of CTLA-4 antagonism has thus opened the possibility for a readily applicable tumor immunotherapy by temporary removal of CTLA-4-mediated inhibition using antagonistic Abs (Egen et al., 2002). Although the mechanisms by which CTLA-4 regulates T cell responses are not completely understood, blocking its activity with an antagonistic or blocking mAb offers a novel approach that holds a promise for immunotherapy. A set of corresponding U.S. patents such as U.S. Pat. No. 5,811,097, U.S. Pat. No. 5,855,887, U.S. Pat. No. 6,051,227, U.S. Pat. No. 6,207,156 and U.S. Pat. No. 7,229,628, illustrates approach of CTLA-4 blockade to strongly enhance antitumor responses has been highly regarded for the treatment potentials.
The anti-CTLA4 blocking mAbs, e.g., clone BNI3 (Steiner et al., 1999) (commercially available from BD Pharmingen) and clone AS33 (Antibody Solutions, Mountain View, Calif.), are often in use to detect sCTLA-4 in biological fluid (Oaks and Hallett, 2000) and to purify Tregs from activated peripheral blood (Birebent, et al., 2004). However, these may not be the best available strategy. Structural analyses have shown that the human CTLA-4 protein is composed of disulfide-linked homodimers of extracellular immunoglobulin variable (IgV) domains, each domain consisting of two layered β-sheets with ten strands (A, A′, B, C, C′, C″, D, E, F and G) that form three complementarity determining region (CDR)-like regions (Schwartz et al., 2001; Stamper et al., 2001). Together with one mutational study (Peach et al., 1994), these two structural studies have independently pointed out that CDR1-like (the B-C loop) and CDR3-like (the F-G loop) regions in CTLA-4 directly bind endogenous B7 ligands (CD80 and CD86), whereas CDR2's responsibility is very trivial if there is any. Therefore, although in the initial publications there is no definite information to describe the CTLA-4 epitope on which the blocking mAbs bind, antagonistic effects and the subsequent enhancement on T-cell activation may be mediated by mAb competition that results from specific binding with amino acid residues on or close to a room encompassing CDR1 and CDR3. Thus uses of blocking mAbs in pair or in combination with endogenous B7 ligands provide a possible limitation caused by steric hindrances.